Abstract

The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α-ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt carboxy-terminal tails inserting into pockets of the α-ring. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit. Here we report that the base subassembly of the Saccharomyces cerevisiae proteasome, which includes the Rpt ring, forms a high-affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6 and Rpn14. Chaperone-mediated dissociation was abrogated by a non-hydrolysable ATP analogue, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α-pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3-pocket. Although the Rpt6 tail is not visualized within an α-pocket in mature proteasomes, it inserts into the α2/α3-pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme.

a, Purified base (160 nM) and CP (80 nM) were incubated with or without Rpn14, Nas6, and Hsm3 (trio, 1.6 μM each), and resolved via native PAGE. Above, in-gel peptidase assay (0.02% SDS); below, Coomassie stain. For input protein see .b, Base (5 nM) and CP (2nM) were challenged with chaperone trio (amounts in molar excess of base; ATP at 2 mM). In this and all real-time experiments, LLVY-AMC hydrolysis is expressed as relative fluorescence units (r.f.u.) and experiments were performed in triplicate with traces combined for presentation.c, Native gel analysis of base-CP formation as in a, following addition of chaperones to base (160 nM) singly or in combination at 10-fold molar excess of base.d, A yeast Rpt hexamer model was built using with the hexameric P97 D1 domain structure as template (see ). This model was fit into the EM map of yeast Rpt hexamer. Relative positions of Hsm3 (red) and Nas6 (yellow) on the Rpt ring (blue) were assessed by superimposing Hsm3-Rpt1C and Nas6-Rpt3C structures onto the Rpt ring model that had been fit into the EM map. A clipped view of the Rpt ring with bound chaperones and CP (green) is presented. Areas of overlap highlight steric clashes between chaperones and CP.

3D reconstruction of base-CP complex reveals an asymmetric interaction between Rpts and the α ring of the CP

a, 3D reconstruction of singly-capped base-CP complex was determined by single particle cryoEM to a resolution of ~10Å (EMD-5617). CP subunits are rendered in different colors as indicated. A difference map was calculated between the original 3D reconstruction and one rotated 180° around the 2-fold CP symmetry axis. The positive difference density (grey) corresponds to base bound to CP. It shows prominent densities from C termini of Rpt6, Rpt2, and Rpt1, which are clustered on one side of the Rpt ring, bound to specific α pockets.b, Each panel shows an α pocket. Thresholds of CP and base densities are set separately but are identical in all panels. C termini of Rpt6, Rpt2, and Rpt1 are seen to insert into α pockets.